A target-triggered dual amplification strategy for sensitive detection of microRNA

• A novel fluorescence biosensor for highly sensitive and selective detection of miRNA was established.
• A dual signal amplification strategy based on single enzyme (DSN) was developed and evaluated.
• DNA/2-OMe-RNA chimeric probe was utilized to connect two rounds of cyclical amplification.
• This strategy can easily be applied to all miRNAs and holds great potential for further application.

The accurate and quantitative analysis of microRNA (miRNA) expression is critical for biomedical research and clinical theranostics. In this study, we report a novel sensor for the sensitive detection of miRNA based on a duplex-specific nuclease (DSN)-assisted dual signal amplification strategy. A chimeric probe (DNA/2-OMe-RNA) that consists of a miRNA recognition DNA sequence and a Taqman probe hybridization RNA sequence (2′-O-methyl RNA) was designed and synthesized. One molecule of target miRNA can trigger cyclical cleavage of the chimeric probes to produce 2′-O-methyl RNA by DSN in the first round of amplification. The 2′-O-methyl RNA molecules can subsequently hybridize with Taqman probes and initiate the second round of cyclical amplification to generate detectable fluorescence by DSN. The proposed strategy exhibits high specificity in discriminating cognate miRNAs, and the dual signal transduction process enables the detection of miRNA concentrations as low as 7.3 fM. We further applied this assay to miRNA quantification in cancer cells to confirm its applicability. The present study provides a sensitive, specific and simple method for miRNA detection and holds great potential for further application in biomedical research and in the clinical laboratory.